The goal of this project is to develop a new technology to systematically delineate disease mechanisms of genetic mutations at the protein level. Identification of the large number of disease-associated mutations has shifted the research challenge of defining underlying disease mechanisms from genomics to functional proteomics. One of the examples is tubby-like protein 1 (Tulp1) that has 23 known mutations associated with retinal degeneration. Although various technologies of functional proteomics have been used to identify a few Tulp1-binding proteins, three challenges are: (a) how to sensitively identify less abundant protein-protein interactions (PPIs);(b) how to quantify binding activity of the entire PPI dataset;and (c) how to systematically distinguish disease-relevant PPIs. Consequently, disease relevance of Tulp1 PPIs is yet to be defined, and its pathological mechanisms remain elusive. We developed open reading frame phage display (OPD) as a new technology of functional proteomics to complement the existing technologies. Owing to its capacity to enrich clones displaying binding proteins through multi-round selection and amplification, OPD should be more sensitive to identify less abundant PPIs than other technologies that lack protein amplification capacity. Other advantages of OPD include its unique capacity to globally map mutation-specific PPIs by next generation sequencing (NGS) and to quantitatively compare the binding activity of entire PPI datasets to wild-type proteins and their pathogenic mutants for reliable delineation of mutation-specific PPIs. The hypothesis is that OPD is a new technology that can be used for global mapping of functionally-relevant and disease-relevant binding proteins of Tulp1. The specific aims are: (1) To test the working hypothesis that OPD systematically identifies mutation-specific PPIs;(2) to test the working hypothesis that OPD-NGS hybrid technology globally maps total PPIs and mutation-specific PPIs. (3) To test the working hypothesis that OPD reliably delineates mutation- specific and biologically-relevant PPIs in a biochemical pathway. This project will facilitate delineation of Tulp1 disease mechanisms of retinal degeneration. Given its broad applicability, OPD-NGS as the only technology for global mapping of mutation-specific PPIs will advance our understanding of disease mechanisms for many other proteins and their genetic mutations. PUBLIC HEALTH RELEVANCE: The goal of this project is to develop a new technology to systematically delineate disease mechanisms of genetic mutations at the protein level. The new technology will be developed to identify and characterize disease-relevant binding proteins and pathological mechanisms for an eye protein that has 23 known blindness-causing mutations. This technology is applicable to other proteins and their pathogenic mutations. Thus, this study has broad impact on our understanding of disease mechanisms associated with genetic mutations.